Chemicals and Reagents
Sch A (purity ≥ 98%) was purchased from Solarbio Biotechnology Co., Ltd (Beijing, China). MPA (purity ≥ 98%) was purchased from Yuanye Biological Technology Co., Ltd (Shanghai, China). Specific antibodies were purchased from different companies as follows: GAPDH (1:10000; ab181602, Abcam, UK); Bax (1:1000; ab32503, Abcam, UK); ZO-1, Caspase-3 (1:1000; #AF5145, #DF6879, Affinity Bioscience, China), p-ERK, P38, p-P38, JNK, p-JNK (1:500; sc-7383, sc-7972, sc-166182, sc-7345, sc-6254, Santa Cruz Biotechnology, USA), ERK (1:1000; 4695S, Cell Signaling Technology, USA), and Bcl-2, occludin (1:1000; 383309, 502601, ZENBio, China).
Cell culture
The human colon adenocarcinoma cell line Caco-2 was kindly provided by Dr. Shuai Song (The First Affiliated Hospital of Anhui Medical University, China). Cells were routinely cultured in complete Dulbecco's modified Eagle's medium (DMEM) (4.5 g/L glucose) (Gibco, MD, USA) with 10% fetal bovine serum (Gibco, MD, USA) and 1% streptomycin (100 µg/mL)/penicillin (100U/ mL) at 37 ◦C with 5% CO2.
Western blotting
Cells were lysed with RIPA lysis buffer containing 1% protease inhibitor (Beyotime Biotechnology, China). Protein samples were loaded and separated by 10% or 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were then blocked with 5% nonfat dry milk in TBST (10 mM Tris, 150 mM NaCl, and 0.1% Tween 20) buffer for 1h at room temperature. Then the membranes were washed three times with 0.1% T-TBS and each membrane was incubated with primary antibodies overnight at 4 ◦C. After washing with TBST the next day, the membranes were probed with the appropriate secondary peroxidase-conjugated antibody (ZSGBbio, China) for 1 h at room temperature. For all gels, GAPDH was used as the internal standard. The membranes were detected by electrochemiluminescence (Applygen Technologies, Inc. China). Protein bands were quantified and normalized with Image J software.
Cell viability assay
A density of 4 × 103 cells seeded in 96-well plates with complete medium. Every other day, the cells were treated with different concentrations MPA and Sch A for 24 h or 48 h, then assessed for viability by the methyl thiazolyl tetrazolium (MTT) assay. The absorbance was assessed at 490 nm with a microplate spectrophotometer (Bio-Tek, USA). All experiments were repeated at least three times.
Immunofluorescence
The cells were seeded on a 24-well plate. After treating the cells with different concentrations of MPA and Sch A for 24 h, the cells were washed with PBS and fixed in 4% formaldehyde at room temperature for 15 min. It was then rinsed in PBS and permeabilized in 0.5% Triton X-100 for 10 min at room temperature. The cells were rinsed in PBS and then blocked with 1% bovine serum albumin for 30 min at 37 °C. After rinsing with PBS, the cells were incubated overnight with ZO-1 antibody (1:250; Affinity Bioscience, China), occludin antibody (1:100; ZENBio, China) at 4 °C. Then, the secondary antibody was incubated for 1 h in the dark at room temperature. 4', 6 diamine phenlinone (DAPI) was used for nuclear staining. The fluorescence was visualized using a confocal microscope (Zeiss, Germany).
Intracellular reactive oxygen species (ROS) measurement
ROS was measured using a flow cytometry (Beckman Coulter, Brea, CA, USA) and a fluorescence inverted microscope (Olympus Corporation, Japan). Cells were seeded and cultured in 6-well or 24-well plates to 90% confluence, as mentioned before, treated them with MPA and Sch A. The cells for flow cytometry were digested the adherent cells with trypsin to prepare a viable cell suspension (106 cells/mL). Treated with 1 ml of H2DCFDA working solution (H2DCFDA stock solution was diluted with PBS to prepare 5 µM) (KeyGEN BioTECH, China) at 37 °C for 20 min. Then, washed the cells twice with PBS, and immediately detected with flow cytometry or fluorescence inverted microscope.
Superoxide dismutase (SOD) activity analysis and malondialdehyde (MDA) content determination
Caco-2 cells in logarithmic growth phase were inoculated into cell culture flasks, fused and grown for one week, and treated with drugs for 24 h as described above. Washed three times with PBS, added lysate to collect cells, centrifuged at 1000r for 10 min to get the supernatant. BCA protein quantification kit (Beyotime Biotech, China) was used to detect protein content, and the final treatment solution was added to 96-well plates according to the instructions of SOD and MDA assay kit (Jiancheng Bioengineering Institute, China), and the absorbance was detected at wavelengths of 450 nm or 530 nm. Then calculated SOD activity and MDA content according to the calculation formula.
Apoptosis Analysis
After the cell culture was completed, the cells were digested without EDTA, and the cells in the culture medium were collected. Centrifuge at 300g for 5 min in a centrifuge. Discarded the supernatant, added PBS to resuspend and centrifuge, repeated 2 times. Resuspended the cells with 400 μL of AnnexinV binding solution, added 4 μL of AnnexinV-FITC in the dark, and incubated for 15 min in the dark. Then added 7.5 μL of PI staining solution, mixed gently, incubated in the dark for 5 min, and then tested in the flow cytometer.
Statistical analysis
All data were statistically analyzed using Statistical Program for Social Sciences v.26.0 software (SPSS Inc., Chicago, IL, USA). The data was normally distributed and expressed by the mean standard deviation (SD). The difference analysis was carried out by one-way (ANOVA). All experiments were repeated at least three times. P < 0.05 was considered statistically significant.